Inferring Function from Shotgun Sequencing Data

Abstract

Methods are described for detecting genes that encode toxic proteins using maps derived from shotgun libraries by determining the presence of gaps in clone start sites on either side of open reading frames. The method is exemplified by identifying a previously unknown restriction endonuclease gene.

1 . A method for identifying an open reading frame (ORF) encoding a toxic protein, comprising: 
 (a) obtaining an in silico map of a plurality of shotgun clones from a shotgun library aligned on a target DNA sequence;    (b) detecting a gap in the map corresponding to a numerical deficiency in start sites of the shotgun clones in a region such that there is a statistically underrepresented number of clones spanning the ORF; and    (c) determining whether a protein product of the ORF is a toxic protein.    
     
     
         2 . A method according to  claim 1 , wherein the region starts at approximately one end of the ORF and extends away from the ORF.  
     
     
         3 . A method according to  claim 1 , wherein the target DNA fragment is a genome  
     
     
         4 . A method according to  claim 3 , wherein the genome is a selected from a bacterial genome, an archaeal genome and a viral genome.  
     
     
         5 . A method according to  claim 3 , wherein the toxic protein is a restriction endonuclease.  
     
     
         6 . A method according to  claim 3 , wherein the toxic gene is mapped to an ORF adjacent to a methylase.  
     
     
         7 . A method according to  claim 6 , wherein the step of identifying the gene expressing the toxic protein from the ORF further comprises expressing the ORF in vivo or by in vitro translation.  
     
     
         8 . A method for identifying an open reading frame (ORF) encoding a toxic protein, comprising: 
 (a) obtaining an in silico map of shotgun clones from a shotgun library aligned on a target DNA sequence;    (b) detecting a gap in the map corresponding to a lack of start sites of the shotgun clones in a region such that there is a lack of clones spanning the ORF; and    (c) determining whether a protein product of the ORF is a toxic protein.